scholarly journals Stress granule-inducing eukaryotic translation initiation factor 4A inhibitors block influenza A virus replication

2017 ◽  
Author(s):  
Patrick D. Slaine ◽  
Mariel Kleer ◽  
Nathan Smith ◽  
Denys A. Khaperskyy ◽  
Craig McCormick
Keyword(s):  
Protein Synthesis ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Translation Initiation Factor ◽  
Host Protein ◽  
Initiation Factor ◽  
Viral Protein Synthesis ◽  
Infected Cells ◽  
Pateamine A

ABSTRACTEukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5’ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection results in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates the feasibility of targeting core host protein synthesis machinery to prevent viral replication.IMPORTANCEInfluenza A virus (IAV) relies on cellular protein synthesis to decode viral messenger RNAs. Pateamine A and silvestrol are natural products that inactivate an essential protein synthesis protein known as eIF4A. Here we show that IAV is sensitive to these eIF4A inhibitor drugs. Treatment of infected cells with pateamine A or silvestrol prevented synthesis of viral proteins, viral genome replication and release of infectious virions. The irreversible eIF4A inhibitor pateamine A sustained long-term blockade of viral replication, whereas viral protein synthesis quickly resumed after silvestrol was removed from infected cells. Prolonged incubation of either infected or uninfected cells with these drugs induced the programmed cell death cascade called apoptosis. Our findings suggest that core components of the host protein synthesis machinery are viable targets for antiviral drug discovery. The most promising drug candidates should selectively block protein synthesis in infected cells without perturbing bystander uninfected cells.

scholarly journals Paramyxovirus-Induced Shutoff of Host and Viral Protein Synthesis: Role of the P and V Proteins in Limiting PKR Activation

2007 ◽  
Vol 82 (2) ◽  
pp. 828-839 ◽  
Author(s):  
Maria D. Gainey ◽  
Patrick J. Dillon ◽  
Kimberly M. Clark ◽  
Mary J. Manuse ◽  
Griffith D. Parks
Keyword(s):  
Protein Synthesis ◽  
Hela Cells ◽  
Viral Protein ◽  
Time Course ◽  
Translation Initiation Factor ◽  
Host Protein ◽  
Initiation Factor ◽  
Viral Protein Synthesis ◽  
Viral Mrnas ◽  
V Protein

ABSTRACT The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI− mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI− mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI− virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2α. WT SV5 was a poor activator of the eIF-2α kinase protein kinase R (PKR). By contrast, the P/V-CPI− mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI− mutant was restored to ∼50% that of control HeLa cells. By contrast, the rates of P/V-CPI− viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI− mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI− mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.

scholarly journals Host and Viral Translational Mechanisms during Cricket Paralysis Virus Infection

2009 ◽  
Vol 84 (2) ◽  
pp. 1124-1138 ◽  
Author(s):  
Julianne L. Garrey ◽  
Yun-Young Lee ◽  
Hilda H. T. Au ◽  
Martin Bushell ◽  
Eric Jan
Keyword(s):  
Protein Synthesis ◽  
Viral Protein ◽  
Rna Virus ◽  
Virus Production ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
S2 Cells ◽  
Structural Protein ◽  
Viral Protein Synthesis ◽  
Paralysis Virus

ABSTRACT The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2α phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2α by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2α phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2α phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5′ untranslated region (5′UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5′UTR and IGR IRES.

scholarly journals RNase L limits host and viral protein synthesis via inhibition of mRNA export

2021 ◽  
Author(s):  
James M. Burke ◽  
Alison R. Gilchrist ◽  
Sara L. Sawyer ◽  
Roy Parker
Keyword(s):  
Protein Synthesis ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Cytokine Production ◽  
Mrna Export ◽  
Virus Protein ◽  
Rnase L ◽  
Viral Mrna ◽  
Viral Protein Synthesis

AbstractRNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Herein, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L-mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. Importantly, the heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L-mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.One Sentence SummaryRNase L-mediated shutoff of nuclear mRNA export limits viral protein synthesis and regulates antiviral cytokine production.

scholarly journals Inhibition of Ongoing Influenza A Virus Replication Reveals Different Mechanisms of RIG-I Activation

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
GuanQun Liu ◽  
Yao Lu ◽  
Qiang Liu ◽  
Yan Zhou
Keyword(s):  
Protein Synthesis ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Viral Rna ◽  
Chain Elongation ◽  
Type I ◽  
Viral Polymerase ◽  
Viral Protein Synthesis

ABSTRACTPattern recognition receptors provide essential nonself immune surveillance within distinct cellular compartments. Retinoic acid-inducible gene I (RIG-I) is one of the primary cytosolic RNA sensors, with an emerging role in the nucleus. It is involved in the spatiotemporal sensing of influenza A virus (IAV) replication, leading to the induction of type I interferons (IFNs). Nonetheless, the physiological viral ligands activating RIG-I during IAV infection remain underexplored. Other than full-length viral genomes, cellular constraints that impede ongoing viral replication likely potentiate an erroneous viral polymerase generating aberrant viral RNA species with RIG-I-activating potential. Here, we investigate the origins of RIG-I-activating viral RNA under two such constraints. Using chemical inhibitors that inhibit continuous viral protein synthesis, we identify the incoming, but notde novo-synthesized, viral defective interfering (DI) genomes contributing to RIG-I activation. In comparison, deprivation of viral nucleoprotein (NP), the key RNA chain elongation factor for the viral polymerase, leads to the production of aberrant viral RNA species activating RIG-I; however, their nature is likely to be distinct from that of DI RNA. Moreover, RIG-I activation in response to NP deprivation is not adversely affected by expression of the nuclear export protein (NEP), which diminishes the generation of a major subset of aberrant viral RNA but facilitates the accumulation of small viral RNA (svRNA). Overall, our results indicate the existence of fundamentally different mechanisms of RIG-I activation under cellular constraints that impede ongoing IAV replication.IMPORTANCEThe induction of an IFN response by IAV is mainly mediated by the RNA sensor RIG-I. The physiological RIG-I ligands produced during IAV infection are not fully elucidated. Cellular constraints leading to the inhibition of ongoing viral replication likely potentiate an erroneous viral polymerase producing aberrant viral RNA species activating RIG-I. Here, we demonstrate that RIG-I activation during chemical inhibition of continuous viral protein synthesis is attributable to the incoming DI genomes. Erroneous viral replication driven by NP deprivation promotes the generation of RIG-I-activating aberrant viral RNA, but their nature is likely to be distinct from that of DI RNA. Our results thus reveal distinct mechanisms of RIG-I activation by IAV under cellular constraints impeding ongoing viral replication. A better understanding of RIG-I sensing of IAV infection provides insight into the development of novel interventions to combat influenza virus infection.

scholarly journals RNase L limits host and viral protein synthesis via inhibition of mRNA export

2021 ◽  
Vol 7 (23) ◽  
pp. eabh2479
Author(s):  
James M. Burke ◽  
Alison R. Gilchrist ◽  
Sara L. Sawyer ◽  
Roy Parker
Keyword(s):  
Protein Synthesis ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Mrna Export ◽  
Virus Protein ◽  
Rnase L ◽  
Viral Mrna ◽  
Viral Protein Synthesis ◽  
Translation Arrest

RNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Here, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L–mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. The heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L–mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.

scholarly journals Rotavirus Infection Induces the Phosphorylation of eIF2α but Prevents the Formation of Stress Granules

2007 ◽  
Vol 82 (3) ◽  
pp. 1496-1504 ◽  
Author(s):  
Hilda Montero ◽  
Margarito Rojas ◽  
Carlos F. Arias ◽  
Susana López
Keyword(s):  
Protein Synthesis ◽  
Virus Replication ◽  
Viral Protein ◽  
Stress Granules ◽  
Viral Proteins ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Cellular Proteins ◽  
Cell Protein ◽  
Infected Cells

ABSTRACT Early during the infection process, rotavirus causes the shutoff of cell protein synthesis, with the nonstructural viral protein NSP3 playing a vital role in the phenomenon. In this work, we have found that the translation initiation factor 2α (eIF2α) in infected cells becomes phosphorylated early after virus infection and remains in this state throughout the virus replication cycle, leading to a further inhibition of cell protein synthesis. Under these restrictive conditions, however, the viral proteins and some cellular proteins are efficiently translated. The phosphorylation of eIF2α was shown to depend on the synthesis of three viral proteins, VP2, NSP2, and NSP5, since in cells in which the expression of any of these three proteins was knocked down by RNA interference, the translation factor was not phosphorylated. The modification of this factor is, however, not needed for the replication of the virus, since mutant cells that produce a nonphosphorylatable eIF2α sustained virus replication as efficiently as wild-type cells. In uninfected cells, the phosphorylation of eIF2α induces the formation of stress granules, aggregates of stalled translation complexes that prevent the translation of mRNAs. In rotavirus-infected cells, even though eIF2α is phosphorylated these granules are not formed, suggesting that the virus prevents the assembly of these structures to allow the translation of its mRNAs. Under these conditions, some of the cellular proteins that form part of these structures were found to change their intracellular localization, with some of them having dramatic changes, like the poly(A) binding protein, which relocates from the cytoplasm to the nucleus in infected cells, a relocation that depends on the viral protein NSP3.

scholarly journals Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis

2002 ◽  
Vol 76 (3) ◽  
pp. 1206-1212 ◽  
Author(s):  
Mirella Salvatore ◽  
Christopher F. Basler ◽  
Jean-Patrick Parisien ◽  
Curt M. Horvath ◽  
Svetlana Bourmakina ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Expression ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Vero Cells ◽  
Cellular Protein ◽  
Host Protein ◽  
Ns1 Protein ◽  
Host Protein Synthesis

ABSTRACT The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-α/β) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein—lacking approximately half of its carboxy-terminal end—showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.

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